Lipid-modifying enzymes such as phosphoinositide kinases, phosphoinositide phosphatases, phospholipases, etc., which induce structural changes (modifications) of lipid molecules on cell membranes, are enzymes involved in cell signaling and evoke diverse cellular responses due to the changes in actions of these lipid-modifying enzymes accompanied by stimulation to the cells. These lipid-modifying enzymes are targeted by various therapeutic drugs, and screening studies have been widely conducted especially on inhibitors/activators. For example, phosphoinositide 3-kinase γ, which is one of phosphoinositide kinases, has been extensively studied as a target molecule for an anti-inflammatory drug (e.g., Non-Patent Literature 1: T. Rückle et al., Nat. Rev. Drug Discovery, 5, 903 (2006)).
In recent years, it has been discovered that the activities of these lipid-modifying enzymes in cell signaling are regulated through the interactions with proteins present on cell membranes.
Examples of the combination of lipid-modifying enzymes and cell membrane proteins controlling the activities are shown in TABLE 1 (Non-Patent Literature 2: U. Maier et al., JBC, 274, 29311 (1999), Non-Patent Literature 3: Ref: E. Buck and R. R. Iyengar, J. Biol. Chem., 276, 36014 (2001) and Non-Patent Literature 4: N. Wittschureck and S. Offermanns, Physiol. Rev., 85, 1159 (2005)).
TABLE 1Examples of lipid-modifying enzymes andcontrolling cell membrane proteinsLipid-Modifying EnzymeControlling Cell Membrane ProteinPhosphoinositide-3-kinase αTyr-phosphorylated receptorPhosphoinositide-3-kinase βTyr-phosphorylated proteins,G-protein βγ subunitsPhosphoinositide-3-kinase γG-protein βγ subunitsPhosphoinositide-3-kinase δTyr-phosphorylated proteinsPhospholipase C βG-protein αq subunits,G-protein βγ subunitsPhospholipase C γTyr-phosphorylated proteins
Accordingly, drugs to treat diseases by controlling the activities of these lipid-modifying enzymes can directly target their modifying reactions (namely, target the interactions between lipid substrates, ATP, etc. and enzymes) and additionally can target the interactions between these enzymes and cell membrane proteins which control the enzymes. However, such drugs have little been developed so far. Extensive development of drugs is ongoing in, for example, the phosphoinositide 3-kinase γ described above, which act on the enzyme to exhibit an anti-inflammatory action, but these drugs basically bind to the ATP-binding site and directly inhibit the modifying reactions (cf., Non-Patent Literature 1).
One of the reasons is that methods for assaying the interactions between a lipid-modifying enzyme and a cell membrane protein regulating the enzyme are not simple. In other words, to assay for such interactions, a lipid served as substrate should be present in the form of a lipid micelle so that studies to investigate such interactions have all been carried out using lipid micelles. However, in most studies using lipid micelles as substrate, methods to extract lipids resulting from the reaction with a solvent are employed. These methods are unsuitable for screening to find a controlling drug.
For example, the following methods are known for assaying the activities of lipid-modifying enzymes.
To assay the activity of phosphoinositide 3-kinase, conventional methods are used which involve preparing lipid micelles containing molecules served as substrate, such as phosphatidylinositol (PI), phosphatidylinositol [4,5]-diphosphate (PIP2), etc., reacting the lipid micelles using 32P and 33P-ATP, extracting the lipids with an organic solvent and finally separating and quantifying the produced phosphatidylinositol monophosphate (PIP) or phosphatidylinositol [3,4,5]-triphosphate (PIP3) by thin layer chromatography, etc. (e.g., Non-Patent Literature 5: T. M. Bonacci et al., Science, 312, 443 (2006), Non-Patent Literature 6: U. Maier et al., J. Biol. Chem., 274, 29311 (1999) and Non-Patent Literature 7: C. A. Parish et al., Biochemistry, 34, 7722 (1995)).
Alternatively, a simple assay method using the proximity effect is also used, which involves “indirect” detection and quantization of PIP3 produced through the reaction of soluble PIP2 as substrate with non-radioisotope ATP by using as an indicator the ability to compete for binding biotinylated soluble PIP3 to PIP3-binding proteins (e.g., Non-Patent Literature 8: A. Gray et al., Anal. Biochem., 313, 234 (2003), Patent Literature 1: B. E. Drees et al., U.S. Pat. No. 7,067,269 and Patent Literature 2: B. E. Drees et al., US Patent 2005/0009124). However, the substrate is soluble PIP2. Application of this method using a lipid-micellized substrate is not reported and it is considered difficult to apply said method to an assay using lipid micelles as a substrate.
In addition, a method which involves immobilizing PIP2 as substrate on FlashPlate where phospholipids have been immobilized and measuring the uptake of 33P from 33P-ATP (e.g., Non-Patent Literature 9: PerkinElmer Inc., Scientific Poster #H78394), a method which involves binding lipid micelles containing substrate to SPA (scintillation proximity assay) beads through an electrostatic or hydrophobic interaction and similarly measuring the 33P uptake, and the like are reported as simple assay methods using the proximity effect (e.g., Non-Patent Literature 10: GE Healthcare Life Sciences, Inc., Scientific Poster #132). However, since immobilization of PIP2 onto FlashPlate or binding of lipid micelles to SPA beads is based on an electrostatic or hydrophobic interaction, it is easy to envisage the drawbacks that substrate molecules are poorly incorporated, the immobilization or binding is susceptible to surfactant effect, etc. These assay methods are scarcely used for practical applications, which is clear also from very few reports in literatures or the like.
Also, a method for assaying the activity of phospholipase which involves preparing lipid micelles containing 3H-labeled PIP2 and measuring the enzymatically hydrolyzed inositol triphosphate (IP3) left in an aqueous layer through organic solvent extraction is used in general (e.g., Non-Patent Literature 11: B. Yoe-Sik et al., Mol. Pharm., 63, 1043 (2003)).
Furthermore, an assay kit is put in practical use for a simple assay method, which involves preparing lipid micelles containing [3H]-PIP2 having a biotinyl group in the molecule, carrying out the reaction with phospholipase, adding streptavidin-coated SPA beads and determining the level of 3H remained on the lipid micelles without solvent extraction (e.g., Non-Patent Literature 12: GE Healthcare Inc., Product No. TRKQ7040, protocol attached). This method using the compound containing in its molecule the biotin residue and the substrate residue susceptible to the actions of lipid-modifying enzyme might be used for measurement of the activities of other lipid-modifying enzymes than phospholipase, if the substrate residue part is changed. For this purpose, however, it is necessary to synthesize, for each enzyme, a compound with a biotin residue introduced in the molecule, not a readily available substrate for lipid-modifying enzyme, which makes any simple measurement difficult.
The foregoing problems involved in the prior art are summarized below.
(1) In the assay system for screening a controlling substance for lipid-modifying enzymes, evaluation including the interactions with controlling cell membrane proteins can be made only when lipid micelles containing lipids served as a substrate are prepared and used as the substrate. In general, a complicated (low-throughput) assay method for measuring the amount of a substrate changed through the reaction using a radioisotope followed by solvent extraction is used for the evaluation of lipid-modifying enzymes, especially “including interactions with cell membrane proteins to be controlled.”
(2) The following methods are known as homogeneous assay methods using lipid micelles containing lipids which are served as a substrate:
a) for phosphoinositide kinases, a method which comprises immobilizing the micelles to beads or plates using an electrostatic or hydrophobic binding function; and,
b) for phospholipases, a method using a compound containing both the biotin residue and a residue served as an enzyme substrate in the same molecule. However, it is readily expected for the method a) that the immobilizing efficiency is poor or the effects of a surfactant are significant. In the method b), it is necessary to design and synthesize a compound suitable for the enzyme in a creative way. Any of these methods is simply not applicable to a variety of “lipid-modifying enzymes.”
Regarding lipid micelles containing biotinylated lipids such as biotinylated phosphatidylethanolamine, etc., it is known that biotin-binding proteins such as streptavidin, etc. bind onto the micelles (e.g., Non-Patent Literature 13: J. Musti et al., Biochemistry, 40, 14869 (2001)) and the micelles bind to a carrier, where biotin binding proteins such as streptavidin, etc. are immobilized (e.g., Non-Patent Literature 14: B. Peker et al., Biotechnol. Prog., 20, 262 (2004)). There is a report on, for example, the measurement of the binding function of proteins bound onto the micelles by applying this method (e.g., Non-Patent Literature 15: W. S. Davidson et al., J. Lipid Res., 47, 440 (2006)).    [Non-Patent Literature 1] T. Rückle et al., Nat. Rev. Drug Discovery, 5, 903 (2006)    [Non-Patent Literature 2] U. Maier et al., JBC, 274, 29311 (1999)    [Non-Patent Literature 3] Ref: E. Buck and R. R. Iyengar, J. Biol. Chem., 276, 36014 (2001)    [Non-Patent Literature 4] N. Wittschureck and S. Offermanns, Physiol. Rev., 85, 1159 (2005)    [Non-Patent Literature 5] T. M. Bonacci et al., Science, 312, 443 (2006)    [Non-Patent Literature 6] U. Maier et al., J. Biol. Chem., 274, 29311 (1999)    [Non-Patent Literature 7] C. A. Parish et al., Biochemistry, 34, 7722 (1995)    [Non-Patent Literature 8] A. Gray et al., Anal. Biochem., 313, 234 (2003)    [Patent Literature 1] B. E. Drees et al., U.S. Pat. No. 7,067,269    [Patent Literature 2] B. E. Drees et al., US patent 2005/0009124    [Non-Patent Literature 9] PerkinElmer Inc., Scientific Poster #H78394    [Non-Patent Literature 10] GE Healthcare Life Sciences, Inc., Scientific Poster #132    [Non-Patent Literature 11] B. Yoe-Sik et al., Mol. Pharm., 63, 1043 (2003)    [Non-Patent Literature 12] GE Healthcare Life Sciences, Inc., Product No. TRKQ7040, Attached Protocol    [Non-Patent Literature 13] J. Musti et al., Biochemistry, 40, 14869 (2001)    [Non-Patent Literature 14] B. Peker et al., Biotechnol. Prog., 20, 262 (2004)    [Non-Patent Literature 15] W. S. Davidson et al., J. Lipid Res., 47, 440 (2006)